Cheng Yaping, Li Quanxiao, Kong Yu, Huang Ailing, Yang Zhenlin, Ying Tianlei, Wu Yanling
Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS) and Shanghai Institute of Infectious Disease and Biosecurity, School of Basic Medical Sciences, Fudan University, 130 Dong'an Road, Shanghai 200032, China.
Shanghai Engineering Research Center for Synthetic Immunology and Shanghai Key Laboratory of Lung Inflammation and Injury, Fudan University, 130 Dong'an Road, Shanghai 200032, China.
J Leukoc Biol. 2025 Aug 5;117(8). doi: 10.1093/jleuko/qiaf107.
Natural killer (NK) cells represent a promising effector population for tumor immunotherapy. Conventional NK cell engagers (NKCEs), primarily targeting CD16a, show efficacy but face limitations due to receptor polymorphisms and off-target toxicity. Here, we report the development and characterization of a novel trispecific NK cell engager (TriKE) simultaneously targeting the activating receptor NKp30 and the tumor-associated antigen HER2, integrated with a modified interleukin-15 (IL-15 N72D) fused to the IL-15Rα sushi domain (IL-15RαSu) to enhance NK cell proliferation and persistence. Protein expression and affinity analyses confirmed the proper formation of the fusion protein with high-affinity binding to NKp30, HER2, and IL-15 receptor components. Flow cytometry demonstrated dose-dependent binding of the TriKE to primary human NK cells and HER2+ tumor cells. Functionally, the TriKE induced significantly greater NK cell activation, as measured by CD69 expression, compared with a bispecific engager lacking IL-15. Importantly, cytotoxicity assays revealed superior NK-mediated killing of HER2+ tumor cells upon prolonged exposure, highlighting the immunostimulatory effect of the IL-15 moiety. These results establish the αNKp30 TriKE as a potent platform to redirect NK cytotoxicity against HER2+ tumors, combining targeted receptor engagement with cytokine-driven activation to enhance NK cell-based cancer immunotherapy.
自然杀伤（NK）细胞是肿瘤免疫治疗中一种很有前景的效应细胞群体。传统的NK细胞衔接器（NKCEs）主要靶向CD16a，虽显示出疗效，但由于受体多态性和脱靶毒性而面临局限性。在此，我们报告了一种新型三特异性NK细胞衔接器（TriKE）的开发和特性，该衔接器同时靶向激活受体NKp30和肿瘤相关抗原HER2，并与融合了IL-15Rα寿司结构域（IL-15RαSu）的修饰白细胞介素-15（IL-15 N72D）整合，以增强NK细胞的增殖和持久性。蛋白质表达和亲和力分析证实了融合蛋白的正确形成，其与NKp30、HER2和IL-15受体成分具有高亲和力结合。流式细胞术表明TriKE与原代人NK细胞和HER2+肿瘤细胞呈剂量依赖性结合。在功能上，与缺乏IL-15的双特异性衔接器相比，通过CD69表达测量，TriKE诱导的NK细胞活化明显更强。重要的是，细胞毒性试验显示，长时间暴露后，NK对HER2+肿瘤细胞的杀伤作用更强，突出了IL-15部分的免疫刺激作用。这些结果确立了αNKp30 TriKE作为一个有效的平台，可将NK细胞的细胞毒性重定向至HER2+肿瘤，将靶向受体结合与细胞因子驱动的激活相结合，以增强基于NK细胞的癌症免疫治疗。
